SUMMARY This application requests continued funding for the TRiP, a highly successful in vivo Drosophila functional genomics platform at Harvard Medical School. With this platform, which to date we have used to generate more than 11,000 RNAi fly stocks for the research community, we propose to build and develop a new resource based on CRISPR technology that will provide powerful, versatile and transformative tools for gene activation, repression and genome engineering. The goals of this new resource are to: 1. Enable systematic GOF screens, which are not doable with existing reagents today; 2. Complement existing RNAi collections; and 3. Enable genome engineering and creation of mosaics. Specifically, we propose to build a genome scale collection of 9,000 transgenic lines for selected genes each expressing constitutively two sgRNAs that are complementary to sequences upstream of the TSS of each gene. Cas9 proteins with dead nuclease activity (dCas9), fused to either transcriptional activators (dCas9-a) or repressors (dCas9-i), can be used for gene activation and repression in cells expressing the sgRNAs, respectively. When combined with the Gal4/UAS system, dCas9-a allows tissue specific overexpression and misexpression experiments, and dCas9- i generates knockdown phenotypes, thus complementing existing RNAi resources. Further, as targeting wild type Cas9 in the soma can be used to generate mutant mosaics, and in the germline can be used to generate small deletions or introduce specific modifications by homologous recombination in the presence of appropriate donor templates for homologous recombination, the lines will be useful for engineering the 5' end of the genes and generate deletions of the region upstream of the TSS. In addition, we will build a set of Gal4>?Cas9? stocks that will allow expression of the various forms of Cas9 (Cas9, dCas9-a, and dCas9-i) in specific tissues. We will characterize the resource to some extent by comparing side-by-side the efficacy of knock down generated by RNAi and dCas9-i, and evaluate the level of overexpression obtained with dCas9-a. Finally, we will curate the information on the quality of individual RNAi and sgRNA lines and collect the information in our RSVP database to include data on the functional quality of CRISPR transgenic lines data. Altogether, this tremendous resource, which will be distributed to the community by the Bloomington Drosophila Stock Center, will create for Drosophila a situation in which researchers can easily access fly stocks useful to `dial down,' `dial up,' or in some cases turn off completely any gene covered by the collection, in any given stage or tissue (as facilitated using the large collection of available Gal4 drivers), and as a result, facilitate a near limitless array of single-gene and multiplexed genetic experiments, as well as facilitate further analysis and integration of the resulting phenotypic data.